Lesson 7: Hemoglobin - Allosteric Modifiers Introduction to Enzymes Flashcards
positive allosteric effector: O2
positive because binding O2 to one site increases affinity at another site
negative allosteric effectors: H+, BPG, and CO2
heterotropic negative allosteric modifiers — these all decrease a subunit’s affinity for O2
what happens if we mutate 2 His residues in central cavity to Ala
(Hb)
if ala binds, means that 2,3 - BPG binds less (needs a basic, positive environment) … if you bind less of it it’ll shift to R state (graph pushed left)
- still cooperativity from the other oxygen binding
- between stripped Hb and Blood Hb
what happens if we mutate 4 His residues in central canal to Ala?
same as 2 mutations, but even more so to the left
- N+ terminals will bind some BPG still, but the amount overall is reduced, more R dominant reaciton
- between stripped Hb and Blood Hb
what if we kept 4 His residues and added 2 more Lys to the central cavity
Lys is basic –> adding more positive charge denity, more 2,3-BPG stabilization for T-state, in front of the Blood Hb
where can the homodimer in the lecture show cooperative oxygen binding
salt bridges between N and C termini, multiple subunits
will the homodimer exhibit a Bohr effect
yes - HIS 13 will function like HIS 146 in Hb
- deprotonate which increases oxygen bonding
what is a bohr effect
changing O2 binding affinity by changing pH
would the addition of BPG have an effect on O2 bindingt (to the central cavity that does not have a lot of positive charge density)
not likely – requires a LARGE amount of positive charge density in the central cavity
what would be the effect of a mutation which replaced aspartic acid 85 with a lysine
most likely increase O2 binding
——- charge charge respulsion, cause the helix to spread apart and O2 can get into the canal
how does CO2 modulate Hb binding affinity
through the HCO3- buffering and carbonic anhydrase
CO2 buffering
- CO2 diffuses from the tissues to the rbc
- carbonic anhydrase causes rxn to quickly yield HCO3- and H+ (a majority of CO2 is carried through the vascular system in the form of HCO3-)
- since H+ is being released, it stimulates O2 to be released into the tissues
CO2 alone helps shift () and () O2 transfer
R –> T and increase
BPG alone helps shift () and () O2 transfer
R –> T and increase
combined, BPG and CO2+….
most efficiently shift R–> T and increase O2 transfer
Carbonic Anhydrase
CO2 + H2 <-> H2CO3 <-> H+ and HCO-
- increase CO2 means increase H+
- increase H+ shifts
R –> O2 dissociates
CO2 decreases what
Hb affinity for O2 because of carbonic acid buffering sytem
where are a lot of the pathological substitution mutations structured near
oxygen binding sites
Hiroshima
- B146 (HC3)
- His –> Asp
- disrupts salt bridge in deoxy state
Suresnes
- A141 (HC3)
- Arg –> His
- eliinates bond between Arg 141 and Asn 126 in deoxy state
Hiroshima and suresnes both favor
both favor R state because they destabilize the T-state (“deoxy”)
would ppl with Hb suresnes and Hb hiroshima be sigmoidal
yes – we know because they are still alive meaning that they must have some level of cooperactivity
enzymes allow for
- increased reaction rates (10^3 - 10^19 times)
- “mild” reaction conditions (i.e. physiological, temp, pH)
- great specificity: both substrates consumed and products produced
- coordinate control: reactions can be turned on and off by modulating activity of enzyme
1
oxidoreductase
- oxidation/reduction reactions
ex: oxidases/dehydrogenase
2
transferace
- transfer of functional groups
ex: kinasdes, transaminases
3
hydrolase
- formation of 2 products by hydrolyzing a substrate
ex: peptides, lipases
4
lyase
- cleavage of C-C, C-O, C-N and other bonds by means other than hydrolysis or oxidation
ex: decarboxylases, carboxylases
5
isomerase
- intramolecular rearangements, transfer of groups within molecules
ex: mutases, isomerases
6
ligase
- formation of C-C, C-O, C-S, or C-N bonds using ATP leverage
ex: synthetases
do enzymes change Keq
no
- they cannot change delta G
do enzymes alter the k of the reaction
yes!
relationship between Keq and delta G
as Keq increases, delta G gets more Negative (think: spontaneous)
where does the rate increase come from
the lower activation energy barrier
what do catalysis to
lower the amount of energy required to reach the transition state (activation energy)
is there a change in delta G when a catalyst use
no – there is no change betwen the delta G of the ground state reactants and the ground state products
comparison of rate enhancement by heat vs decrease in activation energy
- at high T, a large number of molecules possess the threshold energy to get over the activation energy barrier
- at low T, only a small number of molecules possess the threshold energy to get over the activation energy barrier
comparison of rate enhancement by heat vs decrease in activation energy continued…
- lowering activation energy increases the number of molecules that possess the threshold energy to get over the activation energy barrier
what does lowering the value of delta G ++ do
increase the number of molecules with sufficient energy to attain the transition state
proximity and orientation
- enzymes bine substrates with geometric and electrostatic complementary
- bring reacting functional groups in close proximity for chemistry to occur
- enzyme maximizes weak interactions between the E and S (aka energy that is released in the form of heat = binding energy) This binding energy propels the enthalpy driven conformational changes in the enzyme
why is the chemistry rate limiting
highest activation energy in catalyzed reaction
ES –> EP
preferential transition state binding
- enzymes preferentially stabilize substrates at or near the transition state
what would happen to delta G if the enzyme stabilized the substrate
the mazimum stabilization of S decreases energy of ES complex below energy of P
– very stable structure –> hard to form products
- catalyzed activation energy barrier would be incredibly large , endergonic rxn
in this model the catalyzed activation energy of delta G ++ cat, is the sum of the uncatalyzed rxn, delta G ++ uncat + contrimuted delta G magnetic interactions
enzyme stabilizing the reaction state
- increases concentration of molecules in transition state
- limits ES -> E + S increases rate of ES-> P
how do we know that enzymes preferentially stabilize transition states
Transition State Analogs
- stable molecules that mimic the proposed transition state
- these molecules bind tightly to the active site
enzyme adenosine deaminase
- binds the transition state analog 6-hydroxy-1,6-dihydropurine 10 x than substrate or prouce
general acid base chemistry
- certain amino acids of the ability to perform acid-base chemistry
catalytic functional groups
His, Asp, Glu, Lys, Arg
^^^^^ normal charged groups
Ser, Thr
^^^^^^ possess OH
Tyr, Cys
covalent catalysis
- amino side chains form a transient covalent bond with substrate
- this describes the specific, transient, covalent bond between E and S –> there will be other bonds being made or broken
electrostatic catalyis
not acid/base - enzyme side chains, components of the peptide bond, and N- and C- termini, can stabilize charged intermediates
ex:
- His 57 R-group stabilizes lone pair on amide N through electrostatic interactions
- Amide portin of enzyme Ser 195 and Zgly 193 stabilize oxyanion through electrostatic interactions
metal ion catalysis
metal ions can stabilize charged groups, carry electrons, or promote S binding
catalytic triad of chymotrypsin: Ser 195 and Adp 102 and His 57
Ser 195 and Asp 102 and His 57 establish H-bond network that facilitates reactivity
- when the molecule folds – these 3 R groups are right next to eachother…. H-bonding!
chymotropsin:
hydrolyzes peptide bonds C-terminal to aromatics
— large substrate binding pocket accommodates aromatic residues such as tyrosine
chymotropsin: step 1
polypeptide substrate binds to enzyme active site
chymotropsin: step 2
Acid/base catalysis and covalent catalysis
– His 57 removes a proton from ser 195, which allows a nucleohilic attack by the serine oxygen on the carbonyl carbon of the peptide
chymotropsin: step 3
ser 195 side chain: covalent bond with substrate carbonyl
electrostatic stabilization of oxyanion
chymotropsin: step 4
acid/base catlysis: His 57 acts as base
electrostatic catalysis: amide protons stabilize oxyanion
chymotropsin: step 5
acid/base catalysis: His 57 acts as an acid oxyanion “breaks down”
chymotropsin: step 6
oxyanion “breaks down” yields product 2 (amino-terminal fragment) and funcitonal catalytic triad)
role of Mg 2+
stabilizing (-) charge on carboxylate
– electrostaic stabilization
